Mapping of the Cladosporium fulvum resistance gene Cf-16, a major gene involved in leaf mold disease in tomato

Tomato (Solanum lycopersicum) is widely cultivated and consumed worldwide. Tomato leaf mold, caused by Cladosporium fulvum, is one of the most devastating diseases in tomato production. At present, some tomato leaf mold resistance (Cf series) genes used in production gradually lose resistance due to the continuous and rapid differentiation of C. fulvum physiological races. The Cf-16 gene derived from the “Ontario7816” tomato cultivar has shown effective resistance in field trials for many years, but few studies have reported on the mapping of the Cf-16 gene, which has not been cloned, limiting its utilization in tomato breeding. Here, we mapped Cf-16 using a novel comprehensive strategy including bulk segregation analysis (BSA), genome resequencing and SSR molecular markers. A genetic analysis revealed that Cf-16 resistance in “Ontario7816” is controlled by one major dominant locus. The Cf-16 gene was mapped in a region of 2.6 cM at chromosome 6 between two markers, namely, TGS447 and TES312, by using an F2 population from a cross between the resistant cultivar “Ontario7816” and susceptible line “Moneymaker.” Two nucleotide-binding-site-leucine-rich repeat (NBS-LRR) resistance genes, namely, XM_004240667.3 and XM_010323727.1, were identified in this interval. They are strong candidates for the Cf-16 gene. The mapping of Cf-16 may speed up its utilization for breeding resistant tomato varieties and represents an important step forward in our understanding of the mechanism underlying resistance to tomato leaf mold.


Introduction
Tomato (Solanum lycopersicum L.) is a global vegetable cultivated and consumed worldside that has outstanding nutritional. Because of its rich flavor, it is also often used as a fruit (Kim et al., 2017). Leaf mold disease caused by Cladosporium fulvum is considered to be one of the most devastating diseases in tomato crops and has threatened tomatogrowing areas worldwide, especially for tomato grown in protected areas. This pathogen reduces both fruit yield and quality and sometimes even kills tomato plants ( Thomma et al., 2005;Mesarich et al., 2014). In the early stages, tomato leaf mold disease symptoms appear as OPEN ACCESS EDITED BY irregular or oval light yellow chlorotic spots on the backs of the leaves. As the disease progresses, the affected leaves grow graybrown or dark-brown velvety mold layers. When the disease is severe, the leaves senesce, wither and fall off prematurely (Zhao et al., 2022). However, it is increasingly difficult to control this disease in tomato-growing regions around the world. Thus, breeding for tomato leaf mold resistance will provide an effective and environmentally friendly alternative to chemical control. C. fulvum is the causal organism of tomato leaf mold, a fungal disease first described by Cooke (1883). The differentiation rate of C. fulvum physiological races is very rapid, and new races are continuously isolated and identified (Enya et al., 2009;Iida et al., 2010;Li et al., 2015). Thus, the resistance effectiveness of some tomato leaf mold resistance (Cf series) genes used in breeding production has gradually been undermined. Therefore, the mining, cloning and application of new Cf genes have become particularly critical.
DNA markers have been widely used in identifying and mapping plant disease resistance genes. In recent years, singlenucleotide polymorphism (SNP) markers have been widely used to identify markers associated with important traits, including tomato disease resistance Liu et al., 2019). Bulked segregant analysis (BSA) can rapidly detect specific genes or genomic regions. In particular, the combination of BSA and genome resequencing accelerates the cloning of genes responsible for important traits (Michelmore et al., 1991;Zou et al., 2016). Moreover, Kompetitive allele-specific PCR (KASP) markers converted from special SNPs are more flexible and efficient than the original SNP markers for use in genotyping and marker-assisted selection (MAS) Yang et al., 2022). However, simple sequence repeat (SSR) markers are still used to study traits of interest by linkage analysis (Rui et al., 2017).
In this study, we explored the genetic characteristics of the Cf-16 gene by developing a large F 2 population and BC 1 populations. Notably, this is the first time that BSA combined with genome resequencing technology and molecular markers has been used for mapping the tomato Cladosporium fulvum resistance gene Cf-16. This study will be valuable for Cf-16 cloning and resistance breeding in tomato.

Materials and methods
Plant materials and C. fulvum inoculation Resistant female "Ontario7816" (P 1 , kindly provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science), whose genome contain the Cf-16 gene, was crossed with the susceptible male "Moneymaker" (P 2 , kindly provided by the Tomato Genetic Resource Center, LA2706). The F 1 individuals were then self-crossed to harvest F 2 seeds, and the BC 1 plants were generated by backcrossing the F 1 plants with "Ontario7816" and "Moneymaker." All plants were grown in the greenhouse under favorable conditions. Ten C. fulvum physiological races (1. At the 5-6 leaf stage, all P 1 , P 2 , F 1 , F 2 , and BC 1 P 2 plants were inoculated with C. fulvum race 1.2.3.4., which is a predominant physiological race in Heilongjiang Province, China. The plants were assessed for disease severity at 15 days postinoculation. Inoculation and assessment of disease severity ratings were performed as described by Xue et al. (2017). The plants were visually assessed for the severity of symptoms on a scale of 0-9 points. Plants with a disease index of 0-3 were regarded as resistant, and those with a score of 5-9 were regarded as susceptible.

DNA extraction, resequencing and association analysis
After inoculation, the F 2 plants were used for genetic analysis and bulked segregant analysis (BSA). The parents (P 1 and P 2 ) and the F 2 lines were prepared for genome resequencing and molecular marker detection, respectively. The resistant pool (F 2 R-pool, 25 resistant plants) and the susceptible pool (F 2 S-pool, 25 susceptible plants) were built by screening resistant and susceptible plants from the 726 F 2 individuals. The cetyltrimethylammonium bromide (CTAB) method was used for DNA extraction from young leaves, including those of the parents and the F2 lines. Bulked DNA samples were also subjected to the CTAB method by mixing equal amounts of DNA at a final concentration of 200 mg (Allen et al., 2006).
The resequencing and associated analysis were performed by BGI Tech (Shenzhen, China). The samples were constructed to generate 200-300 bp small fragment libraries for upsequencing on BGISEQ. The raw data were filtered to obtain clean data by the BGI in-house filter SOAPnuke. Subsequently, the clean data were aligned to the S. lycopersicum genome sequence (ftp://ftp.ncbi.nlm.nih.gov/ genomes/all/GCF/000/188/115/GCF_000188115.3_SL2.50) using Burrows-Wheeler Aligner (Li and Durbin, 2010). SNP and InDel detection processes were performed with Genome Analysis Toolkit (GATK) (Li et al., 2009), and we used an analysis tool developed by BGI to perform annotation and classification. The SNP-index method was used for the association analysis. The SNP index values of the resistant and susceptible pools were calculated using the resistant female "Ontario7816" as a reference.
Frontiers in Genetics frontiersin.org

SSR molecular marker analysis
Based on the candidate region of the association analysis, a total of 58 SSR markers from the Sol Genomics Network (SGN, http:// solgenomics.net/) database were screened (Supplementary Table  S1). SSR molecular markers were performed using the parents, F 1 and 303 F 2 individuals. Genetic linkage mapping was performed by JoinMap4 software (Ooijen and Van, 2006). Based on the electrophoresis results of 8% non-denaturing polyacrylamide gels.

General strategy used for mapping the Cf-16 gene
The general strategy of mapping the Cf-16 gene in "Ontario7816" is illustrated in Supplementary Figure S1. To determine the inheritance pattern of Cf-16 resistance in "Ontario7816" first, 726 F 2 individuals and 106 BC 1 P 2 individuals were planted and inoculated with C. fulvum race 1.2.3.4. The segregation ratios of resistant and susceptible phenotypes were recorded and further examined by the chi-square test. Next, to explore the rough position of the Cf-16 gene, bulked segregation analysis (BSA) combined with parental resequencing was performed. To further map the Cf-16 gene, 303 F 2 individuals were planted and further screened with 58 SSR markers from the SGN (Supplementary Figure S1).

Resistance and genetic analysis of the Cf-16 gene
To identify the resistance potential of the Cf-16 gene, we tested the resistance response of "Ontario7816" and "Moneymaker" to ten different races of C. fulvum (Table 1). "Moneymaker" showed susceptibility to all these physiological races of C. fulvum, while "Ontario7816" demonstrated resistance to all these races. These findings indicate that "Ontario7816" has highly resistant to C. fulvum and is suitable for future breeding work for disease resistance.
Then, we performed a disease assay on the two parents, F 1 , F 2 and BC 1 P 2 individuals at 15 days postinoculation with the predominant physiological race 1.2.3.4 in Heilongjiang Province (Figure 1). "Ontario7816" and F 1 plants were resistant to C. fulvum race 1.2.3.4, while "Moneymaker" plants were susceptible. Chisquare analysis showed that the segregation ratio of resistant and susceptible individuals of the F 2 population was 3:1. The resistant and susceptible BC 1 P 2 plants segregated according to the expected ratio of 1:1 (Table 2). This result confirms that the Cf-16 gene confers resistance via a single dominant gene.

Parental genome sequencing and SNPindex association analysis
A total of 208.96 Gb clean data were obtained by Beijing Genomics Institute (BGI) sequencing, including 109.72 Gb from the parents and 99.26 Gb from the resistant and susceptible pools, all reads were of high quality (92.85% > Q30 > 90.45%) and with a stable GC content (36.28% > GC > 35.32%). The effective depths for the parents and the two F 2 pools were between 56.23 and 67.16. The quality of the sequencing is high and the data are reliable and could be used for subsequent analysis (Table 3).
Based on the resequencing results, we calculated the SNP index values of the two mixed pools using the resistant parent "Ontario7816" as the reference. The Cf-16 gene was localized on tomato chromosome 6 by ΔSNP index analysis (Figure 2). Further analysis revealed an SNP imbalance between 11 and 38 Mb on chromosome 6, while the ΔSNP index of this region was greater than the threshold value at the 99% confidence level. Therefore, 11-38 Mb of chromosome 6 is the preliminary localization interval of the Cf-16 gene for leaf mold resistance in tomato. Based on the SNP index results, 58 SSR markers were used for polymorphic screening of the two parents and F 1 generation to further map the Cf-16 gene (Figure 3). Subsequently, SSR marker analysis was performed on 303 F 2 generations. Linkage mapping was performed using JoinMap 4.0 software based on the phenotypes and marker types of the F 2 generation (Figure 4). The results showed that   Coverage_rate, the ratio of the sequence to the entire genome; Map_reads_rate, the ratio of the number of reads to the reference genome to the number of reads of clean data; Effective_depth, the ratio of the number of bases aligned to the reference genome to the size of the effective genome (N is not included in the reference sequence).
Frontiers in Genetics frontiersin.org the most closely linked markers to the Cf-16 gene were TGS447 and TES312, both with a genetic distance of 1.3 cM. The positions of these two markers on chromosome 6 were 25.21 Mb and 28.84 Mb, respectively, and the physical distance of this candidate interval was 3.63 Mb.

Analysis of candidate genes
Based on the candidate interval of the SNP index and SSR marker analysis, we found that 71 genes were distributed in the DNA fragment associated to the resistance gene Cf-16. Meanwhile, the number of nonsynonymous SNPs between the two parents in this region was 139. The 71 genes in the candidate interval were subjected to further functional annotation and structural analysis. Based on the typical structural features of the cloned tomato leaf mold resistance Cf gene encoding LRR-TM, 2 candidate genes were screened out, XM_004240667.3 and XM_010323727.1.

Discussion
In recent years, tomato leaf mold has become probably the disease that damage tomato production the most. An effective way to control the disease is to breed resistance to leaf mold. However, some tomato leaf mold resistance (Cf series) genes used available hybrids using currently in farms have gradually lost resistance, due to the continuous and rapid differentiation of C. fulvum

FIGURE 3
Screening of some SSR polymorphic markers.
Frontiers in Genetics frontiersin.org physiological races. It was found that the application of the Cf-4 gene in production was overcome by C. fulvum physiological races such as 1.2.4 and 1.2.3.4. Subsequently, the application of Cf-5 and Cf-9, which are commonly used in breeding, was gradually limited by the appearance and identification of physiological races 1. 2.3.4.5, 1.2.3.4.9, 2.5, and 2.4.5 (Li et al., 2015;Zhao et al., 2016). It is crucial to understand the differentiation of C. fulvum physiological races and to characterize the resistance range of new Cf genes before undertaking breeding efforts for disease resistance. The Cf-16 gene derived from the resistant material "Ontario7816" has shown effective resistance in field trials for many years. This study shows that "Ontario7816" has highly resistant to all the identified physiological races of C. fulvum. This result also indicates that this gene is a valuable resource against C. fulvum breeding. Therefore, mapping, cloning, and characterization of this gene may speed up its use in marker-assisted selection for resistance breeding and may have critical implications for our understanding of the resistance mechanism against tomato leaf mold disease. Previous studies have shown that most Cf genes are inherited dominant single genes. In this study, the genetic analysis indicated that the segregation ratios between resistant and susceptible plants in both the F 2 and BC 1 P 2 populations were in accordance with Mendel's law of segregation. The present results clarify that the Cf-16 gene confers resistance via a single dominant gene. At the same time, it also paves the way for the next localization of Cf-16.
In this study, we mapped the Cf-16 gene for the first time into a 2.6 cM region on tomato chromosome 6 between two markers, TGS447 and TES312, using BSA combined with parental resequencing and SSR molecular markers. By functional annotation and structural analysis of the genes in the candidate Frontiers in Genetics frontiersin.org region, we ultimately predicted two candidate genes, XM_ 004240667.3 (LRR receptor-like serine-/threonine-protein kinase At1g51880) and XM_010323727.1 (LRR receptor-like serine-/ threonine-protein kinase RFK1 isoform X1. LRR receptor-like serine-/threonine-protein kinase), the largest class of receptor-like protein kinase (RLK), has an LRR structure and transmembrane region composed of conserved amino acid sequences (Man et al., 2020). The protein structures encoded by the cloned Cf genes Cf-2, Cf-4, Cf-5, and Cf-9 all have the typical extracellular LRR domain, transmembrane domain and a short cytoplasmic domain, and the different numbers of LRRs are responsible for different Cf genes recognizing different pathogen physiological races (Kruijt et al., 2005). The two genes that were ultimately predicted in this study may have similar structures to Cf genes that have been cloned, so they are identified as candidate genes for Cf-16. However, it is essential to conduct expression pattern analysis and further functional verification of these two candidate genes.

Conclusion
In this study, we mapped the Cf-16 gene for the first time into a 2.6 cM region on tomato chromosome 6 between two markers, TGS447 and TES312, using F 2 bulked segregant analysis combined with genome resequencing and SSR molecular markers. Furthermore, we screened 2 possible candidate genes, XM_ 004240667.3 and XM_010323727.1, with LRR-TM structures similar to those of the cloned Cf genes according to their annotation information. The location and candidate gene screening of Cf-16 could lay a robust foundation for later cloning of the Cf-16 gene and applications in MAS selection programs.

Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/sra PRJNA937877.